Field of the Invention
This invention relates to methods for measuring and reporting vascularity in a tissue sample; and more particularly, to methods for evaluation of angiogenesis and hypoxia using digital image analysis platforms.
Related Art
The ability to evaluate the vascular structure of tissue is important in many therapeutic areas, both in areas that seek to increase the growth of blood vessels (pro-angiogenic) and areas that seek to decrease or shrink blood vessels (anti-angiogenic). It is important to be able to evaluate the architecture of the blood vasculature, as well as to determine how adequately blood supply, nutrients, and oxygen are being made available to local tissue, and how adequately waste products are being removed.
Angiogenesis is a biological process of generating new blood vessels from pre-existing blood vessels into a tissue or organ. Under normal physiology, angiogenesis is tightly regulated by many angiogenic factors, and switching of the phenotype depends on a net balance between up-regulation of angiogenic stimulators and down-regulation of angiogenic suppressors. Therapeutic areas where interventions serve to modulate angiogenesis include: atherogenesis, arthritis, psoriasis, oncology, corneal neovascularization, and diabetic retinopathy.
Evaluation of angiogenesis therapy requires measuring the changes on the tissue vasculature. This is made difficult in that it must either be studied: (i) non-invasively, using radiologic or other non-destructive imaging modalities; (ii) evaluated with histopathology using tissue sections; or (iii) with lab assays with two-dimensional and three-dimensional cell cultures. Histopathology provides the highest resolution evaluation of actual tissue architecture, but quantitation requires measurements on a thin section of a three-dimensional blood vessel network.
Over many years, manual and semi-automated approaches have been developed to measure the number and morphometry of endothelial stained vessels in tissue histology sections. There are a wide number of antibodies that have been developed against both endothelial cells as well as other cells associated with vasculature (e.g. smooth muscle actin), or specific subsets of developing endothelial cells. These can be stained using either immunohistochemistry (IHC) techniques or immunofluorescent (IF) techniques, either singly or in combination with other multiplex stains.
A manual technique to evaluate vascularity in tissue samples was described in Brem, S., R. Cotran, and J. Folkman, “Tumor angiogenesis: a quantitative method for histologic grading”, Journal of the National Cancer Institute, 1972. 48(2): p. 347-356. The technique provides a subjective composite rating of 0-100 based on vasoproliferation, endothelial cell hyperplasia, and endothelial cytology.
Weidner developed a micro-vessel density (MVD) approach in 1991, as described in Weidner, N, et al., “Tumor angiogenesis and metastasis correlation in invasive breast carcinoma”, New England Journal of Medicine, 1991. 324(1): p. 1-8. In Weidner, the tissue was surveyed with a 4× objective, and the areas with the most vascularization, or “hotspots” were identified. In these areas, the field of view with the highest vascularization was then counted for vessels with a 20× or 40× objective. Then the second highest field of view is counted, and up to ten fields of view are tabulated in this fashion. Thus, these “hotspot” areas, or areas which appear to an observer to have high vascularity, are chosen by the observer and then micro-vessel density is computed. While studies have ranged from 3 to 5 fields of view or more, most studies utilize the average of the three most vascularized fields of view when reporting results. The counting method itself can be made slightly more objective after the hotspot regions of interest are selected by evaluating the fields of view by using a Chalkey grid eyepiece as suggested in Chalkley, H, “Method for the quantitative morphologic analysis of tissues”, Journal of the National Cancer Institute, 1943. 4(1): p. 47-53; and Fox, S B., et al., “Tumor angiogenesis in node-negative breast carcinomas relationship with epidermal growth factor receptor, estrogen receptor, and survival”, Breast cancer research and treatment, 1994. 29(1): p. 109-116. While the methodology overlaps, and both critically depend on evaluating and selecting “hotspots”, the Chalkey method can be considered a correlate for vessel area, while MVD is more of a correlate for vessel density.
The field and number of investigations using methodology related to this approach grew tremendously in the following ten years. A review paper of MVD limited only to breast carcinoma studies illustrates this growth. In 2002, forty-three independent previous studies linking micro-vessel density to clinical outcome in breast cancer were reviewed, and the clinical utility of the method confirmed as a prognostic factor; see Uzzan, B., et al., “Microvessel Density as a Prognostic Factor in Women with Breast Cancer A Systematic Review of the Literature and Meta Analysis”, Cancer research, 2004. 64(9): p. 2941-2955. Factor VIII was used in twenty seven of these studies, CD31 in ten, and CD34 in eight. The majority (thirty-nine of forty-three) included measurement from the technique developed by Weidner, three studies included Chalkey methods, and seven studies utilized image analysis for an area based method. In these publications, the authors stress the need for better standardization in MVD, as there was high degree of variability in the number of fields observed, and the exact methodology of the counting technique.
The extent that a human observer is unreliable in estimating and identifying hotspots is well illustrated from a paper evaluating MVD in breast carcinomas. In these studies, the manual technique from Weidner was followed, with the observer identifying and then counting vessels in order, for what was perceived as the ten vascularized highest microscope fields of view. The first field counted actually contained the greatest number of microvessels in only 20% of the sections. In the apparent highest five fields identified by observer, the highest field of view was only found in these five fields sixty-five percent of the time; see Martin, L., et al., “Examining the technique of angiogenesis assessment in invasive breast cancer”, British journal of cancer, 1997. 76(8): p. 1046.
Multiple researchers have undertaken to use image analysis for removing the observer variability introduced when attempting to identify hotspots. Van der Laak, J et al., “An improved procedure to quantify tumor vascularity using true color image analysis: comparison with the manual hot-spot procedure in a human melanoma xenograft model”, J. Pathol, 1998. 184: p. 136-143, describes a semi-automated technique which acquired all fields of view from a tissue section, and then identified hotspots based on the higher areas of positive endothelial staining. The technique was improved upon with the introduction of image analysis morphology to use number of vessels per field rather than area when choosing hotspots; see Belien, J et al., “Fully automated microvessel counting and hot spot selection by image processing of whole tumor sections in invasive breast cancer”, Journal of clinical pathology, 1999. 52(3): p. 184-192.
Microvessel density, Chalkey counts, and image analysis methods were analyzed in depth and correlations between MVD and Chalkey counts, both relying on the “hot spot” approach, were compared. The use of two versus three fields of view with Chalkey counts was evaluated, and the degree of correlation (r=0.93) was considered high enough that only two fields was recommended as sufficient, although using the top two versus top three fields of view will generally produce slightly higher average values as described in Offersen, B., M Borre, and J. Overgaard, “Quantification of angiogenesis as a prognostic marker in human carcinomas: a critical evaluation of histopathological methods for estimation of vascular density”, European Journal of Cancer, 2003. 39(7): p. 881-890. This publication further evaluated the prognostic ability comparison between MVD and Chalkey in large cohorts of prostate, breast, bladder, and non-small cell lung carcinomas.
More recent studies are more likely to include image analysis based total microvascular area (TVA) along with MVD counts, see Sharma, S., M Sharma, and C. Sarkar, “Morphology of angiogenesis in human cancer: a conceptual overview, histoprognostic perspective and significance of neoangiogenesis”, Histopathology, 2005. 46(5): p. 481-489. Total microvascular area predates digital pathology, and TVA, MVD, and Chalkey counts all use the same approach with selecting hotspots and several fields of view.
Referring to Hansen, S., et al., “Angiogenesis in breast cancer: a comparative study of the observer variability of methods for determining microvessel density”, Laboratory investigation; a journal of technical methods and pathology, 1998. 78(12): p. 1563, microvessel density, vascular area, Chalkey counting, and stereological area of vascular profiles were compared in breast cancer. The authors found highest reproducibility in Chalkey counting and stereology, and recommend Chalkey counting overall. Earlier, microvessel density, Chalkey count, and area-based computer image analysis were compared in breast carcinomas, with the authors recommending Chalkey counts; see Fox, S. B., et al., “Quantitation and prognostic value of breast cancer angiogenesis: comparison of microvessel density, Chalkey count, and computer image analysis”, The Journal of pathology, 1995. 177(3): p. 275-283.
In the analyzed hotspots, some researchers have looked at other image-analysis based measurements of individual vessels. These have included major axis length, minor axis length, perimeter, compactness (perimeter/area), and more esoteric measurements like shape factor and Feret diameter; see Korkolopoulou, P., et al., “Clinicopathologic correlations of bone marrow angiogenesis in chronic myeloid leukemia: a morphometric study”, Leukemia, 2003. 17(1): p. 89-97; and Korkolopoulou, P., et al., “A morphometric study of bone marrow angiogenesis in hairy cell leukemia with clinicopathological correlations”, British journal of hematology, 2003. 122(6): p. 900-910.
One researcher derived five classes of microvessel patterns in breast carcinomas and used these for stratification and prognostic outcome. The five classes were (a) increased, blood-filled capillaries with some clustering in the tumor; (b) small-sized capillaries in the tumor; (c) small-sized capillaries condensing at the periphery of the tumor (d); compressed delicate capillaries in the tumor; and (e) compressed delicate capillaries surrounding the tumor islands; see Safali, M, et al., “A distinct microvascular pattern accompanied by aggressive clinical course in breast carcinomas: A fact or a coincidence?”, Pathology-Research and Practice, 2010. 206(2): p. 93-97.
Researchers have noted that heterogeneity of vascularity, as measured by the coefficients of variation of microvessel density or area in randomly sampled regions, is lower in tumors compared to normal tissues in prostate; see Van Niekerk, C. G., et al., “Computerized whole slide quantification shows increased microvascular density in pT2 prostate cancer as compared to normal prostate tissue”, The Prostate, 2009. 69(1): p. 62-69; and Bigler, S. A., R. E. Deering, and M. K. Brawer, “Comparison of microscopic vascularity in benign and malignant prostate tissue”, Human pathology, 1993. 24(2): p. 220-226. This may be explained by strongly increased levels of angiogenic factors that result in a saturation of the vascular bed. Vessel density may actually exceed metabolic requirements in tumors, and the result is uniform over vascularization; Hlatky, L., P. Hahnfeldt, and J. Folkman, “Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn't tell us”, J Natl Cancer Inst, 2002. 94(12): p. 883-93.
With the introduction of digital pathology, the entire slide is available as a digital image for image analysis. This is a vastly different biological endpoint than the preceding hot spot analyses. The entire tumor section is potentially available for sampling, rather than only high areas of vascularity. Area-based algorithms have been developed initially for Automated Cellular Imaging Systems (Chromovision), followed by object-based counting by Aperio; see Potts, S. J., et al. “Performance of a novel automated microvessel analysis algorithm across whole slide digital images”, Toxicologic Pathology. 2009, and more recently, object-based counting by other image analysis vendors like Definiens and Visiopharm. Where the literature has been studying in detail the intra-technique differences within hotspots (e.g. MVD versus Chalkey versus TVA) or more recently intra-technique differences between whole slide analysis (vessel counting versus vessel areas), there are no known studies that have asked whether hotspots themselves are a better technique versus overall vascularity with whole slide analysis.
The main endpoint used in MVD has been the number of vessels per square millimeter of tissue section. There are both theoretical and experimental problems with this endpoint. When one considers microvessel density from a stereological viewpoint, recognizing that a two-dimensional tissue section is only one sample from the three dimensional tumor, a number of theoretical problems present themselves. Anything observed on a section should be considered a profile, rather than the actual object. Recording the number of vessel profiles per area is not a measurement with roots in reality. Thicker or thinner sections, under or over staining, higher or lower cellularity in the sample, will all effect this endpoint. One violates all stereological considerations when trying to extrapolate this vessels per area measurement to volume, the best that the statistic can be used for is to compare the effect of one treatment with another, or before and after treatments, rather than as an absolute physical observation.
Experimentally, the difficulty with vessel densities is the ability to adequately number vessels with image analysis. In tumors with limited vascularity consisting of only small microcapillaries, it may be possibly, but as vascularity increases, it becomes difficult for the pathologist (and especially the computer) to determine which vessel profiles should be part of only one vessel. Many researchers resort to an area measurement to overcome this problem, the area of vessel profiles/area of tissue.
One should also return to the question of the end purpose when developing an analytical method. Is the goal to record how many vessels are in a given tissue, or is the goal to evaluate what percentage of the tumor or tissue is accessible to the vascular network? The named applicant has looked at the addition of a perimeter statistic on vessels, as a possible better correlate with oxygenation than number of vessels, but this requires a high degree of computer accuracy in identifying individual vessels. This assumes that large, non-oxygenating vessels are removed (an important software quality control technique recommended by the applicant) and also still suffers from the challenge of adequately assigning endothelial cells to vessel counts.
The existing microvessel density techniques reviewed above each suffers from the difficult of computationally assigning cells to vessels. This difficulty has resulted in limited usage of these techniques in clinical samples. Another disadvantage is that these techniques report the number or area of vessels, when biologically what may be more appropriate is the percentage of a given cell type that is actually near a vessel.